Barnes Rebecca J, Leung Kam Tin, Schraft Heidi, Ulanova Marina
Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, ON P7B5E1, Canada.
Can J Microbiol. 2008 Jan;54(1):48-57. doi: 10.1139/w07-118.
Analysis of bacterial interactions with host cells using multiple techniques is essential for studies on microbial pathogenesis and for the development of new antimicrobial therapies. Pseudomonas aeruginosa is an important opportunistic pathogen that can cause severe, often life-threatening pulmonary infections in individuals with impaired host defense mechanisms. Using a mini-Tn7 transposon delivery system, we have chromosomally labelled the strain P. aeruginosa PAK with a green fluorescent protein gene (gfp) and tested PAKgfp as a research tool for studies of bacteria-host interactions. We were able to reliably and rapidly measure the interactions of PAKgfp with A549 human lung epithelial cells by using flow cytometry, a fluorometric microplate reader-based assay, and fluorescence microscopy. With these analytical tools, we have demonstrated the adhesion of PAKgfp to the extracellular matrix protein fibronectin and the involvement of fibronectin in PAKgfp-A549 cell interactions. PAKgfp can be successfully used to explore the effects of various pharmacological compounds on P. aeruginosa - host cell interactions in both in vitro and in vivo systems, with potentially important medical applications.
使用多种技术分析细菌与宿主细胞的相互作用对于微生物发病机制研究和新型抗菌疗法的开发至关重要。铜绿假单胞菌是一种重要的机会致病菌，可在宿主防御机制受损的个体中引起严重的、往往危及生命的肺部感染。我们使用mini-Tn7转座子递送系统，用绿色荧光蛋白基因（gfp）对铜绿假单胞菌PAK菌株进行了染色体标记，并测试了PAKgfp作为研究细菌-宿主相互作用的研究工具。我们能够通过流式细胞术、基于荧光微孔板读数器的检测方法和荧光显微镜，可靠且快速地测量PAKgfp与A549人肺上皮细胞的相互作用。借助这些分析工具，我们证明了PAKgfp与细胞外基质蛋白纤连蛋白的粘附以及纤连蛋白在PAKgfp-A549细胞相互作用中的作用。PAKgfp可成功用于探索各种药理化合物在体外和体内系统中对铜绿假单胞菌-宿主细胞相互作用的影响，具有潜在的重要医学应用价值。
